Coexistence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes among clinical Pseudomonas aeruginosa isolates in Egypt

Background Data about the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) production in P. aeruginosa compared to the Enterobacteriaceae family is limited. The availability of limited therapeutic options raises alarming concerns about the treatment of multidrug-resistant P. aeruginosa. This study aimed to assess the presence of PMQR and ESBL genes among P. aeruginosa strains. Methods Fifty-six P. aeruginosa strains were isolated from 330 patients with different clinical infections. Phenotypically fluoroquinolone-resistant isolates were tested by PCR for the presence of six PMQR genes. Then, blaTEM, blaSHV, and blaCTX-M type ESBL genes were screened to study the co-existence of different resistance determinants. Results Overall, 22/56 (39.3%) of the studied P. aeruginosa isolates were phenotypically resistant to fluoroquinolones. PMQR-producing P. aeruginosa isolates were identified in 20 isolates (90.9%). The acc(6')-Ib-cr was the most prevalent PMQR gene (77.3%). The qnr genes occurred in 72.7%, with the predominance of the qnrA gene at 54.5%, followed by the qnrS gene at 27.3%, then qnrB and qnrC at 22.7%. The qepA was not detected in any isolate. The acc(6')-Ib-cr was associated with qnr genes in 65% of positive PMQR isolates. Significant differences between the fluoroquinolone-resistant and fluoroquinolone-susceptible isolates in terms of the antibiotic resistance rates of amikacin, imipenem, and cefepime (P value < 0.0001) were found. The ESBL genes were detected in 52% of cephalosporin-resistant P. aeruginosa isolates. The most frequent ESBL gene was blaCTX-M (76.9%), followed by blaTEM (46.2%). No isolates carried the blaSHV gene. The acc(6')-Ib-cr gene showed the highest association with ESBL genes, followed by the qnrA gene. The correlation matrix of the detected PMQR and ESBL genes indicated overall positive correlations. The strongest and most highly significant correlation was between qnrA and acc(6')-Ib-cr (r = 0.602) and between qnrA and blaCTX-M (r = 0.519). Conclusion A high prevalence of PMQR genes among the phenotypic fluoroquinolone-resistant P. aeruginosa isolates was detected, with the co-carriage of different PMQR genes. The most frequent PMQR was the acc(6')-Ib-cr gene. Co-existence between PMQR and ESBL genes was found, with 75% of PMQR-positive isolates carrying at least one ESBL gene. A high and significant correlation between the ESBL and PMQR genes was detected. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03319-z.


Introduction
Pseudomonas aeruginosa (P.aeruginosa) is an opportunistic gram-negative bacterium that can inhabit moist environments in healthcare settings and colonize about 50% of hospitalized patients [1].Strains of P. aeruginosa can survive on minimal nutritional requirements and adapt to a variety of physical conditions, allowing this organism to persist in both hospital settings and the community [2].P. aeruginosa is one of the top-listed bacteria causing 10-11% of nosocomial infections, such as urinary tract infections (UTI), surgical site infections, burn wound infections, pneumonia, and bloodstream infections [1].Recently, P. aeruginosa has been considered one of the most challenging pathogens for drug resistance.The World Health Organisation (WHO) classified P. aeruginosa on the critical priority list of pathogens that need urgent development of new antibiotics to treat their infections [3].
Heavy use of broad-spectrum antibiotics in the last decades led to the emergence of multiple drug-resistant (MDR) strains of P. aeruginosa, resulting in an increased length of hospital stay, increased healthcare costs, and high morbidity and mortality rates.A high prevalence of MDR P. aeruginosa was reported in Egypt and neighboring countries due to improper use of antimicrobial agents because of the negative population's attitudes and lack of policies for antibiotic prescription [4].
Fluoroquinolones are potent synthetic antimicrobial agents that are active against a wide range of gramnegative and gram-positive bacteria.They are classified, according to their discovery history and their antibacterial properties, into four generations.Second-and thirdgeneration drugs (ciprofloxacin and levofloxacin) are commonly used in clinical practice.The rapid global rise in the prevalence of fluoroquinolone-resistant strains has become a major concern in the past few years due to the heavy use of quinolones in human, veterinary, and agricultural medicine [5].
There are two mechanisms mediating fluoroquinolone resistance.The first is chromosomal mutations in genes encoding DNA gyrase and topoisomerase IV enzymes that are the quinolone targets.Before 30 years, the only known resistance mechanisms to quinolone resistance were chromosomally mediated.The second mechanism is plasmid-mediated quinolone resistance (PMQR), which appeared in 1998 [6].PMQR has been increasingly reported among different clinical strains in most parts of the world in the past decade.Generally, the genes responsible for PMQR have three mechanisms: (1) Quinolone resistance (qnr) genes encode proteins that block quinolones by target modification.Seven qnr families have been found in clinical isolates (qnrA, qnrB, qnrS, qnrC, qnrD, qnrE, and qnrVC) [7].(2) The aminoglycoside acetyltransferase (aac(6')-Ib-cr) gene encodes an enzyme that modifies fluoroquinolones and aminoglycosides through acetylation and reduces drug activity [8].(3) Efflux pump genes: quinolone efflux plasmid (qepA) and olaquindox (OqxAB), which excrete hydrophobic fluoroquinolones [9].The resistance pattern to fluoroquinolones has changed among all Enterobacteriaceae and P. aeruginosa due to the dissemination of PMQR genes.Chromosomalmediated resistance is only transmitted vertically from generation to generation, while PMQR genes can be transmitted horizontally as well as vertically, resulting in the rapid global dissemination of these genes [7].
Extended-spectrum β-lactamases (ESBLs) are one of the common mechanisms of resistance in P. aeruginosa.They are also carried on plasmids and can be easily transmitted among different bacteria.ESBLs are serine B-lactamases of class A, one of the four major classes of the Ambler classification of B-lactamases [10].The bla-TEM, blaCTX-M, and blaSHV genes are the most prevalent enzymes in this class A and have been reported in P. aeruginosa strains [11].Despite the increased importance of quinolone resistance and its plasmid-mediated mechanism in the last decade, as well as several reports from Egypt detecting an increased incidence of PMQR genes among Escherichia coli (E.coli) [12] and Klebsiella pneumoniae [13], few studies have addressed this issue with P. aeruginosa in Egypt.The emergence of bacterial strains carrying both ESBL and PMQR genes is now a serious global health threat [14].Due to the increased worldwide occurrence and diversity of PMQR and ESBL genes among P. aeruginosa strains, it is now necessary to identify these genes for the proper initiation of antibiotic therapeutic regimens.To the best of our knowledge, there is a dearth of data regarding the co-existence of PMQR and ESBL genes among P. aeruginosa in Egypt.In this study, we aimed to determine antimicrobial resistance patterns, particularly to fluoroquinolones and cephalosporins, among P. aeruginosa isolates obtained from different clinical specimens from patients admitted to Minia University Hospitals, Egypt.Then molecular screening of resistant isolates for PMQR and ESBL genes was carried out to study the co-existence of different resistance determinants.

Study design
This study was carried out in the Department of Medical Microbiology and Immunology, Faculty of Medicine, Minia University, Egypt.A total of 56 P. aeruginosa isolates were obtained from 330 different clinical specimens of patients admitted to Minia University Hospitals in the period from January 2023 to May 2023.Patients with any history of antibiotic use two weeks before specimen collection were excluded.The study was performed in accordance with the guidelines of the Declaration of Helsinki.Informed consent was obtained from all patients.The study protocol was approved by the Ethics Committee of the Faculty of Medicine of Minia University (Approval No. 631/2023).

Bacterial isolation and identification
A total of 56 non-repetitive P. aeruginosa isolates were recovered from different clinical specimens as follows: urine (n = 31), wound swabs (n = 18), ear swabs (n = 5), and eye swabs (n = 2).Urine samples were collected under complete aseptic precautions in sterile containers, and other clinical specimens were collected aseptically in sterile swabs with transport media.They were transported within 2 h to the microbiology laboratory for immediate examination.Samples were streaked on Mac-Conkey's agar and nutrient agar (Oxoid, UK) and were then purified on cetrimide agar (Scharlu, Spain).After overnight incubation at 37 °C, all isolated colonies were identified morphologically (Gram stain) and by standard biochemical tests (sugar fermentation, catalase +, oxidase +, indole -, methyl red -, citrate +, H 2 S production -, oxidative, and motility) [15].All isolates were identified and confirmed as P. aeruginosa.Then the identified isolates were inoculated in Trypticase soy broth (Oxoid, UK), mixed with sterilized glycerol (20%) after 24 h of incubation, and stored at -20 °C for further testing.

Antibiotic susceptibility testing
The Kirby-Bauer disc diffusion technique was used to identify antimicrobial resistance among P. aeruginosa isolates using Muller-Hinton agar (Oxoid, England) according to Clinical Laboratory Standard Institute (CLSI) guidelines [16].The following commercially available discs (Oxoid, England) were used, including:

DNA extraction
The DNA of 56 Pseudomonas isolates was extracted using a modified boiling technique by the heat shock method [17].After centrifugation of 1.5 ml of overnight bacterial culture at 13,000 rpm for 5 min at 4 °C, the supernatant was removed carefully, and the pellet was suspended in 200 µl of sterile distilled water.The suspended pellet was then put for 15 min in a water bath and immediately shocked by ice-cooling for 10 min.Lastly, centrifugation at 13,000 rpm for 5 min at 4 °C was done, and the supernatant containing genomic DNA was transferred into a new tube for subsequent PCR amplification.The extracted DNA was used immediately or stored at -20˚C until used.

Detection of PMQR genes among the quinolone-resistant isolates
Pseudomonas isolates, phenotypically resistant to any of the tested quinolones, were screened for the following six genes: quinolone resistance genes (qnrA, qnrB, qnrC, and qnrS), the quinolone efflux gene (qepA), and the quinolone modifying enzyme gene (acc(6')-Ib-cr).Single PCR reactions were used for the amplification of each of the PMQR genes.Each PCR reaction was performed in a total volume of 25µL containing 12.5µL of Hot Start Green PCR Master Mix (Applied Biosystems™, USA), 1µL (10 pmol) of each primer, 3µL (300 ng/mL) of DNA template, and 7.5µL of nuclease-free water.The PCR conditions used for amplification of qnr genes (qnrA, qnrB, and qepA) were: Initial denaturation at 95 °C for 15 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and 72 °C for 5 min, and one cycle of final elongation at 72 °C for 5 min.The thermocycling conditions for the other genes (qnrS, qnrC, and acc(6')-Ib-cr) were the same with changed annealing temperatures.The used primer sequences, annealing temperatures, and sizes of amplified fragments of the studied genes [18][19][20][21][22] are listed in Table 1.The PCR products were resolved on 1.5% Agarose gel containing ethidium bromide dye, and the gel was visualized under a UV transilluminator.

Detection of ESBL genes
All P. aeruginosa isolates that were resistant to cephalosporins (50 isolates) were tested by the PCR assay to determine the β-lactamase-encoding genes (blaTEM, blaSHV, and blaCTX-M) using the specific primers [23][24][25].PCR amplification was performed in a thermocycler (Biometra, UNO II, Goettingen, Germany) as follows: 94 °C for 4 min; 35 cycles of 1 min at 94 °C, 1 min at a specific temperature for each primer, and 1 min at 72 °C; and a final extension step of 10 min at 72 °C.The primer sequences and annealing temperatures used are described in Table 1.PCR products were electrophoresed on a 1.5% agarose gel at 100 V, stained with ethidium bromide dye, and finally visualized with a UV transilluminator system.Positive resistant strains from a previous study in our laboratory were obtained and served as the positive control [26].

Statistical analysis
Statistical analysis of clinical and laboratory data of the studied subjects was performed by IBM SPSS software (version 19.0).A P value of < 0.05 was considered as an indication of statistical significance.

Detection of PMQR genes
The frequencies of the studied six PMQR genes among all phenotypic fluoroquinolone-resistant P. aeruginosa isolates are shown in Fig.  3, with a significantly higher rate of the qnrS gene among isolates from ear discharge than urine and wounds (P value = 0.009).

Discussion
The increasing local reports of PMQR genes and their association with ESBLs among Enterobacteriaceae are alarming and can be a major factor in the increasing prevalence of these genes among Pseudomonas strains [12,27].Extensively improper use of broad-spectrum antibiotics in our country has exacerbated this problem.
In the current study, P. aeruginosa isolates were detected in 19.3% of the collected clinical specimens.That was similar to the results reported by Saki et al. [28] and fewer than Farhan et al. [29].Regarding the specimen site, P. aeruginosa was isolated from 31 of 210 (14.3%) urine samples, 18 of 85 (21.2%) wound swabs, five of 20 (25%) ear swabs, and two of 15 (13.3%) eye swabs.Farhan and his colleagues [29] found a similar prevalence among wound swabs (19.5%) and a higher prevalence in urine (20%) and ear swabs (68.4%).Twenty-two of the isolates (39.3%) were resistant to at least one or both tested fluoroquinolones (ciprofloxacin and levofloxacin).This result was like previous local reports [30][31][32] and worldwide results from Poland [33] (39.6%),Brazil [34] (42%), Saudi Arabia [20] (42.4%),Nigeria [35] (35.5%), and China [36] (32%).In contrast to our findings, Saki et al. [28] and Molapour et al. [37] from Iran reported higher resistance of clinical P. aeruginosa isolates to fluoroquinolones.However, Abbas and his colleagues [38] found high susceptibility (> 90%) of P. aeruginosa isolated from urinary tract infections to ciprofloxacin and levofloxacin.This variation in resistance patterns among isolates could be due to differences in geographical regions, environmental conditions, and  antibiotic prescription patterns among different countries.Also, variation in resistance rates in the same country may be attributed to the different infection types and the disease severity among patients, affecting the drug susceptibility of P. aeruginosa isolates.Fluoroquinolones are one of the most common empiric therapeutic regimens for UTI in our country, explaining that more than 50% of P. aeruginosa isolates resistant to fluoroquinolones were from urine.
In the current study, 90.9% of fluoroquinolone-resistant P. aeruginosa isolates were positive for one or more of the PMQR genes.The acc(6')-Ib-cr gene was the most prevalent PMQR gene among isolates (77.3%).The qnr genes were detected in 72.7% of isolates, with the qnrA gene being the most predominant (54.5%), followed by the qnrS gene (27.3%),then qnrB and qnrC (each 22.7%).In agreement with this result, El-Badawy and his colleagues [20] in Saudi Arabia detected PMQR genes in 37 of 39 isolates (94%), but qnrS was the most prevalent (79.5%).Rajaei et al. [39] from Iran agreed with our result that the qnrA gene was the most frequent among qnrpositive isolates.Regarding data about PMQR among P. aeruginosa in Egypt, Abdelmegeed and his colleagues (2017) [40] detected a similar percent of PMQR genes (89%) in Pseudomonas isolates with different frequencies (qnrS 41%, qnrB 22%, qnrA 9%, and acc(6')-Ib-cr 22%).On the other hand, Ali et al. (2018) [41] reported PMQR genes in 42.9% of quinolone-resistant P. aeruginosa, with a predominance of acc(6')-Ib-cr and a low percent of qnr genes (qnrS and qnrA).In an earlier study by Saleh et al. [30], qnr genes (qnrS and qnrB) were encountered in only 4.5% of isolates.In a recent study from Iran, the qnr genes were detected in 37.5% of quinolone-resistant P. aeruginosa isolates, with frequency rates as follows: qnrB (29.2%), qnrA (25.8%), and qnrS (20.8%) [28].However, Venkataramana and his colleagues [42] from India reported that 68.5% of clinical strains of P. aeruginosa harbored PMQR genes.The results of the current study disagree with earlier studies from Iran [37] and Turkey [43] that did not detect any qnr gene in quinolone-resistant P. aeruginosa.The higher prevalence of PMQR genes in our study may indicate their increasing rate and reflect that there is extensive and inappropriate use of fluoroquinolones, resulting in the emergence of more resistant isolates in our hospital settings.
The prevalence of the aac(6')-Ib-cr gene was 77.3%; this result agreed with similar studies conducted in India (77.6%) [42] and KSA (71.8%) [20].High rates of this gene were also detected in several studies among Enterobacteriaceae [12,13] and P. aeruginosa in Egypt [41].The high prevalence of this gene could be attributed to the excessive use of aminoglycosides or hydrophilic fluoroquinolones in our country.The aac (6')-Ib-cr gene plays a major potential role in the emergence of clinical ciprofloxacin resistance.
The efflux pump gene qepA was not detected in any of the studied isolates, as reported in many previous studies in Egypt [30], Saudi Arabia [20], Iran [28], India [42], and Turkey [43].Two isolates of quinolone-resistant P. aeruginosa did not carry any tested PMQR genes.The quinolone resistance of these isolates can be due to chromosomal resistance or another PMQR gene not investigated in our study.
The remarkable result of the current study was the high co-occurrence of PMQR genes among the quinoloneresistant isolates.Association of acc(6')-Ib-cr with qnr genes was detected in 65% of positive PMQR isolates.All qnrS gene-positive isolates carried acc(6')-Ib-cr.Cocarriage of more than one qnr gene with acc(6')-Ib-cr was also found in 45% of isolates carrying PMQR.In accordance with these results, the association of PMQR genes among P. aeruginosa isolates has been reported in several studies from various countries, including KSA [20], India [42], and China [44].Recently, the coexistence of three PMQR genes (aac(6')-Ib-cr, qnrS2, and oqxAB) was also reported on a multiple resistance plasmid in E. coli in China [45].This finding is an alarm for the need for the implementation of strict antibiotic policies and infection control strategies to limit the spread of these emerging strains.Different studies associate the presence of the qnr genes with long hospital stays and increased 30-day mortality [46].In support of this, the poorest clinical response and fatal outcomes in murine models of urinary tract infection and pneumonia due to the presence of PMQR genes have also been reported [47].
The co-carriage of PMQR genes has changed the fluoroquinolone resistance pattern of Enterobacteriaceae and P. aeruginosa.Although the long-established view that PMQR genes produce only low-level resistance to fluoroquinolones which does not exceed the clinical susceptibility level, they can complement the chromosomal resistance mechanism to reach clinical resistance through the selection of higher-level resistance strains [7].The enzyme encoded by the aac(6')-Ib-cr gene facilitates the selection of high ciprofloxacin-resistant chromosomal mutants and converts the low-level resistance of fluoroquinolone mediated by this enzyme to high-level resistance when coexisting with qnr proteins.More importantly, there is evidence for emerging clinical quinolone resistance without topoisomerase mutations in a resistant E. coli strain carrying PMQR genes (qnrS1 and oqxAB, as well as overexpressing other efflux pump genes) [48].Also, reports of PMQR genes, either alone or in combination, mediate ciprofloxacin resistance in Salmonella isolates in the absence of any chromosomal mutations [49].PMQR genes may also result in alterations in the expression levels of specific genes controlling the susceptibility levels of unrelated agents [46].
In the current study, we examined the prevalence of ESBL genes among isolates resistant to cephalosporin.Detection of ESBL production among P. aeruginosa by molecular techniques is more accurate, as AmpC production by isolates may interfere with or hide the detection of ESBLs by phenotypic tests [50].We found that 46.4% of all P. aeruginosa isolates and 52% of cephalosporinresistant P. aeruginosa isolates carry ESBL genes.The blaCTX-M gene was the most frequent ESBL (76.9%), followed by blaTEM (46.2%).No isolates carried blaSHV.Co-carriage of blaTEM and blaCTX-M was found in 23% of isolates carrying ESBL genes.In line with our results, Goudarzi et al. from Iran [51], Nasser et al. from Yemen [52], and Ejaz from Pakistan [53] reported that blaCTX-M was the more prevalent ESBL gene than TEM and SHV types among P. aeruginosa.In contrast to our results, blaTEM was the most detected in West Iran among P. aeruginosa isolates from coronavirus disease-19 patients [54].In an earlier study in Minia, Egypt, Noha et al. (2015) detected blaTEM only in 12.5%, and in agreement with our results, no isolate had the blaSHV gene [55].However, Farhan and his colleagues in 2019 found CTX-M15 among 55.5% of ESBL-producing P. aeruginosa [29].Before one decade, blaTEM and blaSHV were the predominant types of ESBLs, and blaCTX was scarcely detected in a random pattern.Today, alarming dissemination of CTX-M-type enzymes was found to be mainly associated with the emerging E. coli ST131 clone that usually harbors IncF plasmids [10].A high prevalence of this clone was recently reported in Egypt among isolates from UTIs or colonizing intestines [56].This may explain the increasing prevalence of the blaCTX-M gene in recent studies when compared with earlier ones.
PMQR genes have been found on heterogenic plasmids varying in size and incompatibility specificity, explaining the dissemination of this resistance around the world due to the spread of multiple plasmids between different bacterial hosts and multiple independent acquisitions of these plasmids [57].The aac(6')-Ib-cr variant can be carried on plasmids of different incompatibility groups, but most commonly IncF type.IncF plasmids carry a wide range of resistance genes to major classes of antimicrobials.ESBL genes, especially those of the CTX-M type, PMQR genes, and genes encoding aminoglycosidemodifying enzymes (acc(6')-Ib-cr), are the major trait of genes carried by IncF plasmids [58].These plasmids are now considered a pandemic due to their widespread detection in different countries among different bacteria from different sources and origins.IncF plasmid carrying the aac(6')-Ib-cr gene has been reported in P. aeruginosa [59].From this data, IncF plasmids play a major role in the global dissemination of different resistance genes.
Megaplasmids that can encode a range of antimicrobial resistance and virulence genes are now of emerging interest in nosocomial infections associated with P. aeruginosa [60].
The co-existence of PMQR genes with blaCTX-M and blaTEM was found in our study (Table 5).About 75% of PMQR-positive isolates harbored at least one ESBL gene.The acc(6')-Ib-cr gene showed the highest association with ESBL genes (13 isolates), followed by the qnrA gene (11 isolates).Co-carriage of the acc(6')-Ib-cr gene with one or more of the qnr and ESBL genes was highly prevalent, representing half of positive ESBL gene isolates.Most isolates that co-carried PMQR and ESBL genes were isolated from urine (46.7%), followed by wounds and ear discharges.The genes coding for both PMQR and ESBL are usually carried on the same plasmid, with higher chances of transmission among different members of the Enterobacteriaceae family and P. aeruginosa strains.The widespread co-existence of these genes in our study supports the possibility of the presence of circulating plasmids carrying those resistance genes.P. aeruginosa is capable of rapidly developing resistance to antibiotics through molecular evolution.Excessive and inappropriate use of antibiotics in both medicine and animal feedstuffs produced selective pressure, resulting in an increased rate of plasmid-mediated horizontal gene transfer and the spread of antibiotic resistance genes.There is evidence that the subinhibitory concentrations of antibiotics and quorum-sensing inhibitory antibiotics resulted in a significant increase in the conjugative transfer of resistance genes from E. coli to pseudomonas [61].The co-existence of ESBLs and PMQR genes increased multidrug-resistant P. aeruginosa strains, causing serious infections because of restrictions on the antibacterial agent's choice, therapeutic failures, high public health costs, increased morbidity, and mortality.
The limitation of our study was the low sample size.From the results of the antibiogram, we found that carbapenems followed by aminoglycosides showed higher susceptible results than fluoroquinolones and cephalosporins.The antibiotic regimens used to treat P. aeruginosa infections need to be updated.

Table 2
Distribution of antibiotic resistance patterns and ESBL genes between phenotypic fluoroquinolone resistance and sensitive P. aeruginosa isolates

Table 6
Correlation matrix (r2) between the different PMQR and ESBL genotypes